U.S. patent application number 09/974913 was filed with the patent office on 2002-04-18 for method for the visual detection of specific antibodies in human serum by the use of lateral flow assays.
Invention is credited to Ford, Glen M., Hubscher, Thomas T., Ruppenthal, Teri M..
Application Number | 20020045195 09/974913 |
Document ID | / |
Family ID | 24769492 |
Filed Date | 2002-04-18 |
United States Patent
Application |
20020045195 |
Kind Code |
A1 |
Hubscher, Thomas T. ; et
al. |
April 18, 2002 |
Method for the visual detection of specific antibodies in human
serum by the use of lateral flow assays
Abstract
This invention discloses a method and composition for detecting
the presence of class specific antibodies reactive with analytes
such as bacteria, allergens, autoimmune antigens, viral proteins,
and carbohydrates by lateral flow techniques. In one embodiment of
the invention, a test sample obtained from bodily fluids reacts
with a gold labeled antigen. The resulting complex travels across
the membrane, and along the lateral flow strip. Red colored lines
formed in specific locations along the test strip indicate the
presence of class specific antibodies in the test specimen. In
another embodiment of the invention, the lateral flow assay serves
as an immunochromatographic screening test for the detection of
allergen-specific IgE antibodies in human serum. Test sample reacts
with gold labeled anti-IgE antibody. The resulting complex travels
across the membrane where immobilized allergens capture the
allergen specific IgE complex. Colored lines are formed int the
test areas to indicate the presence of allergen-specific IgE
antibodies.
Inventors: |
Hubscher, Thomas T.;
(Gaithersburg, MD) ; Ford, Glen M.; (Gaithersburg,
MD) ; Ruppenthal, Teri M.; (Hedgesville, WV) |
Correspondence
Address: |
Jonathan E. Grant
Suite 210
2120 L Street, N.W.
Washington
DC
20037
US
|
Family ID: |
24769492 |
Appl. No.: |
09/974913 |
Filed: |
October 12, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09974913 |
Oct 12, 2001 |
|
|
|
09689682 |
Oct 13, 2000 |
|
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|
Current U.S.
Class: |
435/7.9 ;
435/287.2; 436/514 |
Current CPC
Class: |
G01N 33/558 20130101;
Y10S 435/97 20130101; Y10S 435/975 20130101; Y10S 436/81 20130101;
Y10S 436/821 20130101; Y10S 436/807 20130101 |
Class at
Publication: |
435/7.9 ;
435/287.2; 436/514 |
International
Class: |
G01N 033/53; G01N
033/542; C12M 001/34; G01N 033/558 |
Claims
What we claim is:
1) A lateral flow immunoassay device for detecting immunoreactions,
said device comprising: a test strip, said test strip comprising: a
sample site for applying a sample comprising biologically active
materials, said biologically active materials selected from the
group consisting of antigens and antibodies; a colorimetric
labeling site for labeling the sample, forming a calorimetric
immuno complex, said colorimetric labeling site comprising a
colorimetric label positioned downstream from said sample site; and
at least one reaction site positioned downstream from said
calorimetric labeling site, said at least one reaction site
comprising reactive biological substances which binds said
colorimetric immuno-complexes when said biologically active
materials of said sample react positively with the biological
substances.
2) The lateral flow immunoassay device of claim 1, wherein said
test strip further comprises a membrane support having a testing
layer on the surface thereof, wherein said membrane support is
comprised of a material selected from the group consisting of
plastic, cardboard, nitrocellulose and combinations thereof.
3) The lateral flow immunoassay device of claim 2, wherein said
testing layer further comprises a sample site to which the sample
is applied.
4) The lateral flow immunoassay device of claim 3, further
comprising a sample pad residing on top of the testing layer, to
which the sample is transferred.
5) The lateral flow immunoassay device of claim 1, wherein said
calorimetric labeling site is a calorimetric labeled analyte to
which the sample is being tested.
6) The lateral flow immunoassay device of claim 5, wherein said
colorimetric label is selected from the group consisting of a metal
sol particle, a dyed labeled microparticle, a fluorescent labeled
microparticle, and combinations thereof.
7) The lateral flow immunoassay device of claim 6, wherein said
metal sol particle may be selected from the group consisting of
platinum, gold, silver, selenium, and copper.
8) The lateral flow immunoassay device of claim 5, wherein said
analyte is selected from the group consisting of antigens,
antibodies, bacteria, viruses, protozoa, parasites, autoimmune
antigens, heat shock proteins, transplantation antigens,
histocompatability antigens, and combinations thereof.
9) The lateral flow immunoassay device of claim 8, wherein said
analyte is selected from the group consisting of H. pylori,
Streptococcus Group A, Streptococcus Group B, Mycobacterium
tuberculosis, Mycoplasma, Chlamydiae, Rickettsiae, Herpes virus,
CMV, Hepatitis A, Hepatitis C, Hepatitis B, Influenza, HIV I, HIV
II, HTLV I & II, Chagus, Toxoplasma, Helminh, Nematodes,
autoimmune antigens, heat shock proteins, transplantation antigens,
histocompatability antigens, and combinations thereof.
10) The lateral flow immunoassay device of claim 1, further
comprising at least one binding site downstream from said
calorimetric or fluorometric labeling site.
11) The lateral flow immunoassay device of claim 10, wherein said
at least one reaction site comprises at least three reaction sites,
said reaction sites comprising: an anti-IgA binding site; an
anti-IgM binding site; and an anti-IgG binding site.
12) The lateral flow immunoassay device of claim 11, further
comprising a control site, said control site containing substances
that readily react with any calorimetric or fluorometric
compound.
13) The lateral flow immunoassay device of claim 11, wherein
anti-Ig immunoglobulin antibodies specific for specific antibodies
are positioned at said anti-Ig binding sites.
14) The lateral flow immunoassay device of claim 13, wherein said
anti-Ig immunoglobulin antibodies are from affinity purification of
immune sera selected from the group consisting of goats, rabbits,
donkeys, sheep, chickens, and other animals.
15) The lateral flow immunoassay device of claim 13, wherein said
anti-Ig immunoglobulin antibodies are monoclonal antibodies
specific for IgM, IgA, IgE, and IgG antibodies.
16) The lateral flow immunoassay device of claim 11, further
comprising substances reactive with IgG.
17) The lateral flow immunoassay device of claim 13, further
comprising substances reactive with IgG, combined with said
antibody.
18) The lateral flow immunoassay device of claim 16, wherein said
substances are selected from the group consisting of protein A,
protein G, lentil lectin, jacalin, concanavilin A, anti-IgG, mannan
binding protein, wheat germ lectin, peanut lectin, avidchrom, and
combinations thereof.
19) The lateral flow immunoassay device of claim 4, further
comprising an IgG reacting substance.
20) The lateral flow immunoassay device of claim 19, wherein said
IgG reacting substance is selected from the group consisting of
protein A, protein G, an antibody to IgG, lentil lectin, jacalin,
concanavilin A, mannan binding protein, wheat germ lectin, peanut
lectin, avidchrom, and combinations thereof.
21) The lateral flow immunoassay device of claim 1, when said
calorimetric labeling site comprises labeled anti-IgE
antibodies.
22) The lateral flow immunoassay device of claim 21, further
comprising a plurality of said reaction sites, each said reaction
site being an allergen site, wherein each allergen site has a
different immobilized allergen.
23) The lateral flow immunoassay device of claim 22, wherein said
immunoassay comprises at least one test strip.
24) The lateral flow immunoassay device of claim 22, wherein said
allergens are selected from the group consisting of pollens, dust
mite allergens, molds, animal epithelium, foods, allergen mixes,
and combinations thereof.
25) The lateral flow immunoassay device of claim 22, wherein said
allergens are immobilized on the test strip by the use of
solubilizing agents.
26) The lateral flow immunoassay device of claim 25, wherein said
solubilizing agents are selected from the group consisting of
sugars and alcohols, which allow for the unfolding of the allergen
protein tertiary structure such that more hydrophobic domains are
exposed allowing increased binding to the membrane, and increased
reactivity to the IgE antibody.
27) A lateral flow immunoassay device for detecting immune
reactants, said device comprising: a test strip, said test strip
comprising: a sample site for applying a sample comprising
antibodies; a calorimetric labeling site for labeling the sample,
forming a calorimetric antibody complex, said calorimetric labeling
site comprising a calorimetric labeled analyte positioned
downstream from said sample site ; and at least four reaction sites
positioned downstream from said calorimetric labeling site, wherein
said there is a reaction site for detecting IgG antibodies, a
reaction site for detecting IgA antibodies, a reaction site for
detecting IgM antibodies, and a control reaction site, wherein each
said reaction site binds said colorimetric immune-antibody
complexes when said antibodies of said sample specific for each
said reaction site are present in said sample, whereupon a colored
line will appear at each reaction site where said antibodies of
said sample have been bound to said colorimetric labeled
analyte.
28) The lateral flow immunoassay device of claim 27, wherein said
calorimetric label is selected from the group consisting of a metal
sol particle, a dyed labeled microparticle, a fluorescent labeled
microparticle, and combinations thereof.
29) The lateral flow immunoassay device of claim 28, wherein said
metal sol particle may be selected from the group consisting of
platinum, gold, silver, selenium, and copper.
30) The lateral flow immunoassay device of claim 27, wherein said
analyte is selected from the group consisting of antigens,
antibodies, bacteria, viruses, protozoa, parasites, autoimmune
antigens, heat shock proteins, transplantation antigens,
histocompatability antigens, and combinations thereof.
31) The lateral flow immunoassay device of claim 30, wherein said
analyte is selected from the group consisting of H. pylori,
Streptococcus Group A, Streptococcus Group B, Mycobacterium,
Tubercle bacillus, Mycoplasma, Chlamydiae, Rickettsiae, Herpes
virus, CMV, Hepatitis A, Hepatitis C, Hepatitis B, Influenza, HIV
I, HIV II, HTLV I & II, Chagus, Toxoplasma, Helminths,
Nematodes, autoimmune antigens, heat shock proteins,
transplantation analytes, histocompatability antigens, and
combinations thereof.
32) The lateral flow immunoassay device of claim 27, further
comprising a control site, said control site containing substances
that readily react with the colored solid phase.
33) The lateral flow immunoassay device of claim 27, wherein
anti-Ig immunoglobulin antibodies specific for specific antibodies
are positioned at said anti-Ig binding sites.
34) The lateral flow immunoassay device of claim 33, wherein said
anti-Ig immunoglobulin antibodies are from affinity purification of
immune sera selected from the group consisting of goats, rabbits,
donkeys, sheep, chickens, and other animals.
35) The lateral flow immunoassay device of claim 33, wherein said
anti-Ig immunoglobulin antibodies are monoclonal antibodies
specific for IgM, IgA, IgE, and IgG antibodies.
36) The lateral flow immunoassay device of claim 33, further
comprising substances reactive with IgG.
37) The lateral flow immunoassay device of claim 33, further
comprising substances reactive with IgG, combined with said
antibody.
38) The lateral flow immunoassay device of claim 36, wherein said
substances are selected from the group consisting of protein A,
protein G, lentil lectin, jacalin, concanavilin A, anti-IgG, mannan
binding protein, wheat germ lectin, peanut lectin, avidchrom, and
combinations thereof.
39) The lateral flow immunoassay device of claim 27, further
comprising an IgG reacting substance at said sample site..
40) The lateral flow immunoassay device of claim 39, wherein said
IgG reacting substance is selected from the group consisting of
protein A, protein G, an antibody to IgG, lentil lectin, jacalin,
concanavilin A, mannan binding protein, wheat germ lectin, peanut
lectin, avidchrom, and combinations thereof
41) A lateral flow immunoassay device for detecting immune
reactants, said device comprising: a test strip, said test strip
comprising: a sample site for applying a sample comprising
antibodies; a colorimetric labeling site for labeling the sample,
forming a calorimetric antibody complex, said colorimetric labeling
site comprising a calorimetric labeled anti-IgE antibody, said
colorimetric labeling site positioned downstream from said sample
site ; and a plurality of reaction sites downstream from said
labeling site, each said reaction site containing a different
allergen such that when IgE antibodies labeled with colorimetric
labeled anti-IgE antibodies come in contact with an antigen to
which the IgE antibodies react, the reaction site will develop a
colored line, indicating a positive response.
42) The lateral flow immunoassay device of claim 41, when said
calorimetric labeling site comprises labeled anti-IgE
antibodies.
43) The lateral flow immunoassay device of claim 41, further
comprising a plurality of said reaction sites, each said reaction
site being an allergen site, wherein each allergen site has a
different immobilized allergen.
44) The lateral flow immunoassay device of claim 41, wherein said
immunoassay comprises at least one test strip.
45) The lateral flow immunoassay device of claim 43, wherein said
allergens are selected from the groups consisting of pollens, dust
mite allergens, molds, animal epithelium, foods, allergen mixes,
and combinations thereof.
46) The lateral flow immunoassay device of claim 43, wherein said
allergens are immobilized on the test strip by the use of
solubilizing agents.
47) The lateral flow immunoassay device of claim 46, wherein said
solubilizing agents are selected from the group consisting of
sugars and alcohols, which allow for the unfolding of the allergen
protein tertiary structure such that more hydrophobic domains are
exposed allowing increased binding to the membrane, and increased
reactivity to the IgE antibody.
48) A lateral flow immunoassay device for detecting immune
reactants, said device comprising: a test strip, said test strip
comprising: a sample site for applying a sample comprising
antibodies; a fluorescent labeling site for labeling the sample,
forming a fluorescent antibody complexes, said fluorometric
labeling site comprising a fluorescent labeled analyte positioned
downstream from said sample site; and at least four reaction sites
positioned downstream from said fluorescent labeling site, wherein
said there is a reaction site for detecting IgG antibodies, a
reaction site for detecting IgA antibodies, a reaction site for
detecting IgM antibodies, and a control reaction site, wherein each
said reaction site binds said fluorescent antibody complexes when
said antibodies of said sample specific for each said reaction site
are present in said sample, whereupon a fluorescent line appears at
each reaction site where said antibodies of said sample have been
bound to said floursescent labeled analyte.
49) The lateral flow immunoassay device of claim 48, wherein said
analyte is selected from the group consisting of antigens,
antibodies, bacteria, viruses, protozoa, parasites, autoimmune
antigens, heat shock proteins, transplantation antigens,
histocompatability antigens, and combinations thereof.
50) The lateral flow immunoassay device of claim 49, wherein said
analyte is selected from the group consisting of H. pylori,
Streptococcus Group A, Streptococcus Group B, Mycobacterium,
Tubercle bacillus, Mycoplasma, Chlamydiae, Rickettsiae, Herpes
virus, CMV, Hepatitis A, Hepatitis C, Hepatitis B, Influenza, HIV
I, HIV II, HTLV I & II, Chagus, Toxoplasma, Helminths,
Nematodes, autoimmune antigens, heat shock proteins,
transplantation antigens, histocompatability antigens, and
combinations thereof.
51) The lateral flow immunoassay device of claim 48, further
comprising a control site, said control site containing substances
that readily react with any fluorescent compound.
52) The lateral flow immunoassay device of claim 48, wherein
anti-Ig immunoglobulin antibodies specific for specific antibodies
are positioned at said anti-Ig binding sites.
53) The lateral flow immunoassay device of claim 52, wherein said
anti-Ig immunoglobulin antibodies are from affinity purification of
immune sera selected from the group consisting of goats, rabbits,
donkeys, sheep, chickens, and other animals.
54) The lateral flow immunoassay device of claim 52, wherein said
anti-Ig immunoglobulin antibodies are monoclonal antibodies
specific for IgM, IgA, IgE, and IgG antibodies.
55) The lateral flow immunoassay device of claim 54, further
comprising substances reactive with IgG.
56) The lateral flow immunoassay device of claim 54, further
comprising substances reactive with IgG, combined with said
antibody.
57) The lateral flow immunoassay device of claim 56, wherein said
substances are selected from the group consisting of protein A,
protein G, lentil lectin, jacalin, concanavilin A, anti-IgG, mannan
binding protein, wheat germ lectin, peanut lectin, avidchrom, and
combinations thereof.
58) The lateral flow immunoassay device of claim 48, further
comprising an IgG reacting substance at said sample site.
59) The lateral flow immunoassay device of claim 58, wherein said
IgG reacting substance is selected from the group consisting of
protein A, protein G, an antibody to IgG, lentil lectin, jacalin,
concanavilin A, mannan binding protein, wheat germ lectin, peanut
lectin, avidchrom, and combinations thereof
60) A lateral flow immunoassay device for detecting
immunoreactants, said device comprising: a test strip, said test
strip comprising: a sample site for applying a sample comprising
antibodies; a fluorescent labeling site for labeling the sample,
forming a fluorescent antibody complex, said fluorescent labeling
site comprising a flouroscent labeled anti-IgE antibody, said
fluorescent labeling site positioned downstream from said sample
site ; and a plurality of reaction sites downstream from said
labeling site, each said reaction site containing a different
allergen such that when IgE antibodies labeled with fluorescent
labeled anti-IgE antibodies come in contact with an antigen to
which the IgE antibodies react, the reaction site will develop a
flourescent line, indicating a positive response.
61) The lateral flow immunoassay device of claim 60, when said
fluorescent labeling site comprises labeled anti-IgE
antibodies.
62) The lateral flow immunoassay device of claim 60, further
comprising a plurality of said reaction sites, each said reaction
site being an allergen site, wherein each allergen site has a
different immobilized allergen.
63) The lateral flow immunoassay device of claim 60, wherein said
immunoassay comprises at least one test strip.
64) The lateral flow immunoassay device of claim 62, wherein said
allergens are selected from the group consisting of pollens, dust
mite allergens, molds, animal epithelium, foods, allergen mixes,
and combinations thereof.
65) The lateral flow immunoassay device of claim 62, wherein said
allergens are immobilized on the test strip by the use of
solubilizing agents.
66) The lateral flow immunoassay device of claim 65, wherein said
solubilizing agents are selected from the group consisting of
sugars and alcohols, which allow for the unfolding of the allergen
protein tertiary structure such that more hydrophobic domains are
exposed allowing increased binding to the membrane, and increased
reactivity to the IgE antibody.
Description
DESCRIPTION
[0001] This following application is a divisional of U.S.
application Ser. No. 09/689,682, filed Oct. 13, 2000.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention discloses a method and composition for
detecting the presence of antibodies in human or animal bodily
fluids (blood, serum, plasma, urine, colostrum, milk, tears, or
saliva) to analytes such as bacteria, Chlamydiae, Rickettsiae,
protozoa, allergens, autoimmune antigens, viral proteins, and
carbohydrates by lateral flow techniques.
[0004] 2. Description of the Prior Art
[0005] Over the years, numerous patents have been issued involving
immuno-chromatographic devices. The standard features of these
devices comprise the following:
[0006] a) A plastic or paper housing allowing the viewing of a
reaction area on a bibulous (lateral flow) strip;
[0007] b) An opening at one end of the housing allowing for the
addition of sample (urine, blood, plasma, serum or bacteria in a
media base);
[0008] c) Bibulous material (the lateral flow strip) having
immobilized specific binding members (analytes) capable of reacting
with antigens or antibodies.
[0009] d) A pad of absorbent bibulous material (the absorbent pad)
enclosed at the end opposite the sample well and used to absorb
transversely flowing sample, buffers and colloids;
[0010] e) A strip of bibulous material used in the sample well end
to initially absorb the sample being applied;
[0011] f) A strip of bibulous material in contact with the sample
well material and the lateral flow strip and containing a dried
colored solid phase reagent, the solid phase coated with proteins
or haptens.
[0012] Two types of chromatographic immunoassays are commonly
described. In the one, proteins or small molecule analytes
contained in human fluids (urine, blood, plasma, serum, and saliva)
are detected. The analytes include hCG, FSH, LH, CKMB, TSH,
troponins, myoglobulin, cancer proteins, viral/bacterial proteins,
haptens, therapeutic drugs, and drugs of abuse.
[0013] In the other chromatographic immunoassay, the analyte being
detected is human antibody specifically reactive with agents such
as viral/bacterial proteins (HIV, Hepatitis A and C, H. pylori,
EBV, Rubella, CMV, HSV, Dengue fever, Lyme, Chagas, TB, Toxoplasma,
autoimmune antigens, etc.) or allergens (pollens, molds,
dust/mites, foods, animal epithelia, etc.). The various analytes
are abbreviated VB for simplified use below. When it comes to
detecting antibody, three formats are typically used:
[0014] 1) The colored solid phase [SP] is coated with proteins or
lectins [protein A, protein G, lentil lectin, jacalin, concanavilin
A, mannan binding protein, wheat germ lectin, peanut lectin and
avidchrom] that react with human IgG antibodies. The solid phase
may be coated with anti-immunoglobulins that specifically react
with IgG, IgM, IgA, or IgE. The bibulous strip would in this case
contain the analyte (VB) of interest to which the specific antibody
contained in the sample reacts.
[0015] 2) The colored solid phase contains the analyte (VB) to
which the human immunoglobulins react. The bibulous strip would in
this case also contain the analyte (VB) of interest to which the
specific antibody contained in the sample reacts.
[0016] 3) The colored solid phase contains the analyte (VB) to
which immunoglobulins react.. The bibulous strip contains proteins
directed against human IgG or total immunoglobulins (protein A,
,protein G, lectins, lentil lectin, jacalin, concanavilin A, mannan
binding protein, wheat germ lectin, peanut lectin and avidchrom or
a mix of antibody to immunoglobulin classes IgG, IgA ,IgM and
IgE).
[0017] U.S. Pat. No. 5,459,041 (Blaser et al.) discloses antigenic
compositions for use in diagnostic kits and the like for detecting
the presence of antibodies specific for Campylobacter pylori,
Samples of bodily fluids, for instance, may be contacted with
immobilized antigen on a solid phase which is then washed and
tested for the occurrence of significant levels of antigen/antibody
complex. Levels exceeding a predetermined positive threshold are
indicative of antibodies to Campylobacter pylori in the sample
tested. Kits employing the antigenic compositions of the invention
preferably include means for detecting the antigen/antibody complex
such as materials and reagents for conducting an enzyme-linked
immunosorbent assay, Western blot technique, ELISA, liposome-based
assay or other known detection tests. The Western blot and ELISA
tests used here are for the detection of IgA and IgG
antibodies.
[0018] U.S. Pat. No. 5,567,594 (Calenoff) discloses a library of
isolated and purified antigens specific for a microorganism is a
set of individual molecules. The library forms antigen-antibody
complexes useful in the context of diagnosing and treating
conditions associated with a specific microorganism such as H.
pylori-induced gastro-duodenal disease. For the antigen-antibody
complexes the antibody in question is an immunoglobulin, which is
IgE if the antigens are allergens. Antigen-antibody complexes with
IgA, IgG and IgM are also useful if the antigen is a bacteria.. By
this multivariate approach, a specific condition is diagnosed with
high sensitivity and specificity by determining whether complexes
form between a specific antigen library and a biological sample
which contains immunoglobulins from an individual. Such libraries
also are useful for immunotherapy. Western blot is used to detect
IgE antibodies. The method requires enzyme conjugates and enzyme
substrates and two wash steps to detect antibodies.
[0019] U.S. Pat. No. 5,420,014 (Cripps et al.) discloses a method
for detecting a current infection by H. pylori in a mammal. The
method comprises contacting a mucous secretion [saliva] from said
mammal with an immobilized antigen component from H. pylori for a
time and under conditions sufficient for an IgG antibody in said
mucous secretion specific to a antigen component to form a complex
therewith and then subjecting said complex to a detecting means
which involves an enzyme conjugate and specific substrate.
[0020] U.S. Pat. No. 6,068,985 (Cripps) discloses a method which
uses saliva to detect IgG in both the Western Blot and ELISA tests.
This detection method requires the use of an enzyme conjugate and
enzyme substrate and two wash steps to detect the antibody.
[0021] U.S. Pat. No. 5,846,751 (Pronovost et al.) discloses a
sensitive and specific antigen preparation for the detection of
Helicobacter pylori in biological samples. The preparation uses a
range of antigens derived from size exclusion chromatography of
detergent-solubilized H. pylori cells and the purified antigen
preparation is coated on the solid phase. Serological assays such
as ELISA, latex agglutination, and rapid EIA assays are used to
detect antibodies to H. pylori. The invention also uses a lateral
flow device to detect total immunoglobulins to H. pylori. In this
case, the H. Pylori antigen is striped on the membrane reaction
area and also coated to the colored solid phase. The antibody in
the sample reacts first with H. pylori gold coated conjugate, and
then travels to the membrane reaction area where it reacts with
striped H. pylori.
[0022] U.S. Pat. No. 5,200,344 (Blaser et al) uses a purified p28kd
protein from H. pylori to detect IgA, IgM and IgG antibody in ELISA
and Western Blot. The test requires conjugate and enzyme substrate
and two wash steps to detect the antibody.
[0023] U.S. Pat. No. 6,060,326 and 5,945,294 (Frank et al.)
discloses methods to detect canine IgE using a canine Fc epsilon
receptor to detect canine IgE antibodies in a biological sample
from a canine.
[0024] U.S. Pat. No. 5,547,833 (Dorval et al.) discloses a radial
flow assay delivery device, and methods of use.
[0025] None of these patents teach or disclose a fast and effective
lateral flow assay test for the testing of multiple-class specific
antibodies. More specifically, no chromatographic immunoassay is
able to distinguish between reactive antibody contained in the
classes of human antibody (IgG, IgA, IgM, IgD and IgE). All devices
to date detect either total immunoglobulins or IgG. The problem of
separating reactivities of antibody class lies in the 10 to 15 fold
excess of IgG class specific antibodies over IgA, IgM, and IgE
class specific antibodies reactive with analyte (VB) in question at
various protein sites (epitopes). If the IgG is allowed to react at
the same time or same rate as other classes of antibody, the IgG
will mask most if not all the analyte (VB) epitopes, thereby
decreasing or eliminating the activity of the IgM, IgA, and IgE
class antibodies to the analyte (VB).
SUMMARY OF THE INVENTION
[0026] The proposed invention allows for antibody class
recognition. In one embodiment of the invention, a lateral flow
immunoassay device distinguishes at least three classes of
antibody. The classes of antibody to be distinguished include IgG,
IgA and IgM. A control line reactive with gold particles is also
present. .
[0027] In another embodiment of the invention, the immunoassay test
strip is modified to allow detection of the IgE class of antibody
to many allergens (VB) coated sequentially on a bibulous strip.
Saturated anti-IgE antibodies coated to colored solid [SP] phase
particles at high concentration are reacted with a controlled
amount of serum to allow for the near complete complexing of
elevated levels of human or other animal IgE. This insures that
little free IgE is left unreacted. Unreacted IgE would inhibit the
reaction with the multiple analytes coated on the bibulous strip.
By capturing most of the IgE on the colored solid phase, sufficient
IgE specific antibody molecules are available to react with the
various allergens (VB) as the reaction front moves transversely
down the strip toward the absorbent pad. This allows for the
detection of many different IgE allergen specific molecules.
[0028] In both embodiments of the inventions, an IgG reacting
protein (which can be protein A, protein G, an antibody to IgG or
lectins such as lentil lectin, jacalin, concanavilin A, mannan
binding protein, wheat germ lectin, peanut lectin and avidchrom) is
added to the sample pad in order to complex the IgG contained in
the sample such that the molecular weight of the IgG complex is
greater than 1.0 million. This large complex travels sufficiently
slower than IgA, IgM, and IgE thereby allowing these antibodies to
react prior to the IgG. After reacting to the antigen coated
colored solid phase, the various reacted complexes are captured on
the bibulous strip coated at three sites with antibody to IgM, IgA
and IgG or a protein/lectin reactive with IgG (protein A, protein
G, lentil lectin, jacalin, concanavilin A, mannan binding protein,
wheat germ lectin, peanut lectin and avidchrom). Thus, the class of
reactive antibody is distinguished.
[0029] In another embodiment of the invention, the colored solid
phase contains proteins that react with IgG, allowing for the
detection of many different analyte specific antibody molecules of
the IgG class. The reagents coated onto the bibulous lateral flow
strip include autoimmune antigens, allergens, Chlamidia,
Rickettsiae, viruses, and bacteria.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] The invention will be better understood and further objects,
characterizing features, details and advantages thereof will appear
more clearly as the following description proceeds with reference
to the accompanying diagrammatic drawings given by way of non
limiting example only illustrating a presently preferred specific
embodiment of the invention.
[0031] FIG. 1 is a perspective view of one embodiment of the
lateral flow immunoassay test;
[0032] FIG. 2 is a perspective view of another embodiment of the
lateral flow immunoassay test;
[0033] FIG. 3 is an exploded view of the lateral flow immunoassay
test;
[0034] FIG. 4 is a side view of the test strip;
[0035] FIG. 5 is a view of the colored particles attached to the
antigen; and
[0036] FIG. 6 is an exploded view of another version of the lateral
flow immunoassay test.
DETAILED DESCRIPTION OF THE INVENTION
[0037] The immunoassay lateral flow test system 1 comprises a
casing 2, preferably plastic, surrounding a test strip 7. On the
top surface of the test strip there is a sample opening 8 and a
test results opening 9 to show the results of the assay. The casing
can take a number of different forms. In FIGS. 3 and 6,, the casing
has a top section 4 or 100, and a bottom section 5 or 101. Within
the top section 4 there is at least one channel 6 into which is
fitted a test strip 7. The test strip 7 preferably has a membrane
support 10. The membrane support 10 may be comprised of plastic,
cardboard, or any other rigid material. On top of the membrane
support 10 is a testing layer 11, preferably made out of
nitrocellulose. On top of the nitrocellulose or testing layer 11
are the areas to which the appropriate reagents or samples are
applied or affixed. The nitrocellulose/testing layer may be affixed
to the membrane support 10.
[0038] At one end of the test strip 7 is the sample site 12 to
which the sample is to be applied. This sample site 12 preferably
has a sample pad 13 residing on top of the testing layer, to which
the sample is transferred. The sample is preferably a bodily fluid.
This fluid may be serum whole blood, plasma, colostrum, milk,
saliva, tears, or urine sample from a human or other animal
species.
[0039] Incorporated in the sample site 12 or sample pad 13, or
downstream from the sample site is the labeled antigen, for which
the serum is being tested.
[0040] The gold particles 14 attached to the antigen or antibody 15
are preferably larger than 20 nm, more preferably in the range of
about 20 to 100 nm, and most preferably in the range of 20 to 40
nm. The gold sol labeled antigens/antibodies 35 are dried and
deposited on the strip 7.
[0041] The metal sol particles to be used in accordance with the
present invention may be prepared by coupling the analyte directly
to the gold particle. Additionally, the labeled component may be
prepared by coupling the analyte to the particle using a
biotin/avidin linkage. In this latter regard, the substance may be
biotinylated and the metal containing particle coated with an
avidin compound. The biotin on the analyte may then be reacted with
the avidin compound on the particle to couple the substance and the
particle together. In another alternative form of the invention,
the labeled component may be prepared by coupling the analyte to a
carrier such as bovine serum albumin (BSA),key hole lymphocyananin
(KLH), or ovalbumin and using this to bind to the metal
particles.
[0042] The metal sol particles to be used in accordance with the
present invention may be prepared by methodologies which are well
known. For instance, the preparation of gold sol particles is
disclosed in an article by G. Frens, Nature, 241, 20-22 (1973).
Additionally, the metal sol particles may be metal or metal
compounds or polymer nuclei coated with metals or metal compounds,
as described in U.S. Pat No. 4,313,734. Other methods well known in
the art may be used to attach the analyte to gold particles. The
methods include but are not limited to covalent coupling and
hydrophobic bonding. The metal sol particles may be made of
platinum, gold, silver, selenium, or copper or any number of metal
compounds which exhibit characteristic colors.
[0043] Similarly, the analyte does not necessarily have to be
attached to a metal sol particle, but may instead be attached to
dyed or fluorescent labeled microparticles such as latex,
polystyrene, dextran, silica, polycarbonate, methylmethacrylates
and carbon.. The metal sol particles, dyed or fluorescent labeled
microparticles should be visible to the naked eye or able to be
read with an appropriate instrument (spectrophotometer, fluorescent
reader, etc. ).
[0044] There are a number of ways in which the gold labeled
antigens 16 may be deposited on the strip 7.
[0045] In an alternative and preferred embodiment, the gold labeled
antigens/antibodies are deposited and dried on a rectangular or
square absorbent pad 16, the pad preferably about
0.25".times.0.25"or less. This absorbent pad 16 is positioned
downstream from where the sample is applied on the strip 7.
[0046] In yet another embodiment of the invention, the analytes may
be attached to microspheres. This has the effect of increasing the
number of reactive sites (epitopes) in a given area. Analytes may
be attached to these alternate solid phases by various
methodologies.
[0047] For instance, reactive microspheres (MX-Covaspheres.RTM. of
diameter 0.5 micrometers or 0.9 micrometers) purchased from Duke
Scientific Corporation, Pal Alto, Calif. 94303, or other suppliers,
may be used to covalently attach analytes. The binding is at the
amino groups of the protein if covalent methodology is used. In
addition, hydrophobic or electrostatic domains in the protein may
be used for passive coating. A suspension of the spheres is mixed
after sonication with the antigens/antibodies in water or in a
phosphate buffer solution, after which they are incubated at room
temperature for 10-75 minutes. The mixture is then centrifuged and
the pellets containing the antigen/antibody-linked microspheres are
suspended in a buffer containing 1-5% wt/volume bovine serum
albumin (BSA) for 1 hour at room temperature. The BSA blocks any
unreacted surfaces of the microspheres. After one more
centrifugation, the spheres are resuspended in buffer (TBS with 5%
BSA) and stored at 4 degrees C. before using.
[0048] The solid phase particles may comprise any one of known,
water dispersable particles, such as, the polystyrene latex
particles disclosed in U.S. Pat. No. 3,088,875. Such solid phase
materials simply consist of suspensions of small, water-insoluble
particles to which antigens/antibodies are able to bind. Suitable
solid phase particles are also disclosed, for example, in U.S. Pat.
Nos. 4,184,849; 4,486,530; and 4,636,479.
[0049] In another embodiment of the invention, the analytes may be
attached to fluorescent microspheres or fluorescent microparticles.
Said fluorescent micropaticles may be purchased from Duke
Scientific, Palo Alta, Calif. 94303 and are listed as Green, Red,
or Blue fluorescent 0.4 micron microspheres (Product Bulletin 93).
They are also available from Molecular Probes, Eugene, Oreg. 97402
and are listed as FluoroSpheres; Blue, Yellow-Green, Nile Red,
Orange, Red, Crimson, Dark Red and Far Red in micron sizes from
0.03 to 5.0. Other manufactures also supply fluorescent
microspheres. Characteristically, fluorescent microspheres
incorporate fluorescent dyes in the solid outer matrix or in the
internal volume of the microsphere. The fluorescent spheres are
typically detected by a fluorescent reader that excites molecules
at one wavelength and detects the emission of fluorescent waves at
another wavelength. For example, Molecular Probes Nile Red
particles excite at 526 nm at emit at 574 nm, the Far Red excites
at 680 nm and emits at 720 nm and the Blue excites at 365 nm and
emits at 430 nm. In a lateral flow format, detection of fluorescent
microparticles requires the use of a reflectance reader with an
appropriate excitation source (HeNe, Argon, tungsten or diode
laser) and an appropriate emission filter for detection. Use of
diode lasers allows for use of detection systems that use low cost
lasers with detection above 600 nm. Most background fluorescence is
from molecules that emit fluorescence below 550 nm.
[0050] Fluorescent microspheres contain surface functional groups
such as carboxylate, sulfate and aldehyde groups, making them
suitable for covalent coupling of proteins and other amine
containing biomolecules. In addition, sulfate, carboxyl and amidine
microspheres are hydrophobic particles that will passively absorb
almost any protein or lectin. Coating is thus similar as for non
fluorescent microspheres (Mx-Covaspheres or other latex
microparticles). A suspension of the fluorescent spheres is mixed
after sonication with the antigens/antibody in water or in a
phosphate buffered solution, after which they are incubated at room
temperature for 10-75 minutes. EDAC (soluble carbodiimide),
succinimidyl esters and isothiocyanates as well as other
crosslinking agents may be used for covalent coupling of proteins
and lectins to the microspheres. After the protein has attached to
the surface of the miroparticles, the mixture is centrifuged and
the pellets containing the antigen or antibody linked to the
fluorescent microparticles are suspended in a buffer containing
1-5% bovine serum albumin for one hour. After one more
centrifugation, the spheres are resuspended in buffer (TBS with 5%
BSA or other appropriate buffers) and stored at 4 degrees C. before
use.
[0051] The solid phase particles useful in connection with the
invention may comprise, for example, particles of latex or of other
support materials such as silica, agarose, glass, polyacrylamides,
polymethyl methacrylates, carboxylate modified latex and Sepharose.
Preferably, the particles will vary in size from about 0.2 microns
to about 10 microns. In particular, useful commercially available
materials include 0.99 micron carboxylate modified latex, cyanogen
bromide activated Sepharose beads (Sigma), fused silica particles
(Ciba Corning, lot #6), isothiocyanate glass (Sigma), Reactogel
25DF (Pierce) and Polybead--carboxylate monodisperse microspheres.
In accordance with the invention, such particles may be coated with
a layer of antigens coupled thereto in a manner known per se in the
art to present the solid phase component.
[0052] In the preferred embodiment, the sample contains antibodies
which will react with the gold labeled antigen, forming an
antigen-antibody complex. The gold-antigen antibody complex begins
to migrate along the test strip.
[0053] Further down the length of the test strip are four binding
sites. The first binding site 18 is preferably to bind IgM. The
second binding site 19 is preferably a site to bind IgA. The third
binding site 20 is for the binding of IgG and the fourth binding
site 22 is for a control. More specifically, each binding site is
in the form of a striped line along the width of the test strip
opening 9. At the site of each binding site, there are anti-Ig
immunoglobulins. For example, class specific antibodies are laid
down on the test strip. For example, a goat anti-human IgM antibody
is laid down at the first binding site 18, goat anti-human IgA
antibody is laid down at the second site 19 and goat anti-human IgG
antibody is laid down at the third binding site 20. At the control
site there is immobilized a protein or substance containing sulfur
residues that readily react with any colloidal gold compound. It
can also be an antibody reactive with the proteins coated on the
gold or microparticles surface. Since the gold or microparticles
conjugate is always in excess of sample reactive antibodies,
sufficient conjugate is available to react with the control line.
The antibodies reactive with IgM, IgA and IgG can be from affinity
purification of immune sera from goats, rabbits, donkeys, sheep,
chickens or other animals. It may also be monoclonal antibodies
directed against IgM, IgA and IgG. The antibodies used are specific
for the heavy chain portion of the IgM, IgA and IgG antibodies.
Substances reactive with IgG (protein A, protein G, lentil lectin,
jacalin, concanavilin A, mannan binding protein, wheat germ lectin,
peanut lectin and avidchrom) may be substituted for the antibody to
IgG or combined with said antibody.
[0054] After the sample has been placed at the sample opening 8,
the sample migrates to the site of the gold sol labeled analytes
pad 16. Analytes specific for the gold sol conjugate will attach
and bind, thus forming gold sol labeled complexes. The gold sol
complex continues to migrate along the length of the lateral flow
strip. Reactive complexes are specifically captured by analyte (VB)
coated on the test strip 7. Migration continues and complexes are
captured on the control line of the test strip 7. Excess fluid is
wicked into the absorbent pad 17.
[0055] The problem of separating reactivities of antibody classes
lies in the 10 to 15 fold excess of IgG over IgA and IgM specific
antibody reactivity with analyte reaction sites. If the IgG is
allowed to react at the same time or rate as other classes of
antibody, the IgG will mask most if not all the analyte epitopes,
thereby decreasing or eliminating the activity of the IgM and IgA
class antibodies to the analyte.
[0056] To solve this problem, an IgG reacting substance ( which can
be, among others, protein A, protein G, an antibody to IgG, lentil
lectin, jacalin, concanavilin A, mannan binding protein, wheat germ
lectin, peanut lectin and avidchrom) is added to the sample pad in
order to complex the IgG such that the molecular weight of the
complex is greater than 1 million. This large complex travels
sufficiently slower than IgA, IgM, and IgE, thereby allowing these
antibodies to react prior to the IgG. After reacting with the
colored solid phase, the various reacted complexes are captured
specifically at three sites by the antibodies to IgM, IgA,, and
IgG, or a substance reactive with IgG (protein A, protein G, lentil
lectin, jacalin, concanavilin A, mannan binding protein, wheat germ
lectin, peanut lectin and avidchrom).
[0057] For example, to determine whether a person has been exposed
to Helicobacter pylori, or to determine if there has been
successful treatment of the disease, a serum sample is tested to
determine whether it contains antibodies to H.pylori. Assuming the
use of gold labels, if lines appear at the binding sites for IgG
and weakly for IgM, then there is only a chronic condition present.
If however, lines appear at the binding sites for IgM and IgA, with
or without IgG, then an active or recent colonizing infection is
occurring. The detection of IgA when combined with a low serum
pepsinogen level is associated with an increased risk of gastric
cancer.
[0058] This test can detect the presence of class specific
antibodies reactive with any bacteria, virus, fungus, irritant, or
protein. Some, but not all of the analytes which can be detected
using this method include Streptococcus Group A, Streptococcus
Group B, Mycobacterium, Mycobacterium tuberculosis, Mycoplasma,
Chlamydiae, Rickettsiae, Herpes virus, CMV, Hepatitis A, Hepatitis
C, Hepatitis B, Influenza, HIV I, HIV II, HTLV I & II, Chagus,
Toxoplasma, Helminh, Nematodes, autoimmune antigens, antibodies to
heat shock proteins, transplantation analytes, histocompatability
analytes, and combinations thereof.
[0059] In another embodiment of the invention, this lateral flow
assay can be used for the visual detection of allergen specific IgE
antibodies in human or animal serum. In this assay the test serum
reacts with a colorimetric (preferably gold) labeled anti-IgE
antibody contained in the colorimetric (preferably dried) gold pad.
16. The resulting complex travels along the test strip to the line
stripped allergen site 24. At the allergen site, there are a
plurality of immobilized allergens 24. Indeed, the immunoassay can
easily test for one or more different allergens, preferably by one
strip 25, two strips (25 and 26) or multiple strips next to each
other. Each strip can contain one or more specific allergen lines.
The common allergens which may be tested include but are not
limited to pollens (Timothy, cultivated rye, birch, alder,
hazelnut, mugwort, English plantain, ragweed, nettle, etc.), dust
allergens (D. farinae, D. pteronyssinus, house dust), molds
(Alternaria tenuis, Aspergillus fum., Cladosporium, Penicillium
not), animal epithelium (Cat epithelium, dog dander, horse dander,
goose feathers) foods (dairy, cereals, nuts, seafoods, legumes and
mixes thereof), inhalant mixes (pollen I (grasses), pollen II
(weed/trees), animal mix, dust mix, mold mix) and combinations
thereof. The allergens are immobilized on the test strip by the use
of solubilizing agents such as sugars and alcohols (sucrose,
mannose, fructose, ethylene glycol, ethanol, methanol, glycerin,
dextrans). The use of sugars and alcohols unfolds the allergen
protein tertiary structure such that more hydrophobic domains are
exposed allowing greater binding to the membrane. In addition,
protein to protein aggregation is reduced through solubilization
allowing individual protein molecules to bind to the nitrocellulose
or nylon membrane.
[0060] Assuming there is a reaction between the complexes of gold
labeled anti IgE antibody and the sample containing IgE antibody
and the allergens, a red line will appear at the site of the
allergen when there is a positive response. The assay validity is
demonstrated by the appearance of a red colored line in the
positive control region of the membrane. The positive control is a
protein or substance containing sulfur residues that readily react
with any colloidal gold compound. It can also be an antibody
reactive with the proteins coated on the gold or microparticles
surface. Since the gold or microparticles conjugate is in excess,
sufficient conjugate is available to react with the control
line.
[0061] Many modifications and variations of the present invention
are possible in light of the above teachings. It is, therefore, to
be understood within the scope of the appended claims the invention
may be protected otherwise than as specifically described.
* * * * *